There have been great advances in molecular biology in recent years in terms of new restriction enzymes, improved DNA cloning methods, improved hybridization probes, and improved hybridization supports: however, little, if any, attention has been paid to preparation of the samples for use in these procedures. That is, little has been done to improve the speed and efficiency of isolation and preparation of nucleic acids, specifically DNA, for use in these procedures.
The classic reference for isolation of high molecular weight DNA is Marmur [Journal of Molecular Biology, Volume 3, 208-218 (1961)]. The process taught by Marmur includes 14 steps, some of which must be repeated to insure completeness. Many of these steps are time consuming and most require a highly skilled technician to assure successful isolation of the DNA. The individual steps are discussed below showing all details provided by the author:
1. Cell lysis
A cellular suspension is first treated with a detergent, sodium dodecyl sulfate, at 60.degree. C. in an attempt to lyse the cells. If lysis is achieved, one proceeds to the next step; if not, an enzyme, lysozyme, is added and allowed to digest the cells for 30-60 minutes. These steps can be reversed but, if they are, the detergent must be added for the subsequent processes to work correctly. This method of cell lysis, while broadly effective, is not universally so. There are organisms with cell walls which are resistant to both lysozyme and detergent treatment (e.g. Streptococcus pyogenes).
2. Deproteinization
The viscous lysed suspension is made 1M in perchlorate and extracted with a chloroform-isoamyl alcohol mixture. This mixture is then centrifuged to form 3 layers, the middle layer containing the proteins. The upper aqueous layer contains the nucleic acids and is removed for further processing.
3. Nucleic acid precipitation
The nucleic acids are precipitated by layering ethanol on top of the aqueous layer and collecting them on a glass rod. The precipitate is drained of excess ethanol by pressing the precipitate against the side of the flask. This step requires a highly skilled technician because, as is the case with all DNA precipitation steps to follow, significant losses of the desired DNA can occur. These losses occur due to incomplete precipitation, redissolution of precipitate during washing, and binding of precipitating DNA to the walls of the vessel.
4. Dissolution of nucleic acids
The precipitate is transferred to dilute saline-citrate buffer and the nucleic acid is redissolved by gentle stirring. Excessive stirring causes shearing of the DNA resulting in low molecular weight DNA being formed and, therefore, great care must be exercised.
5. Deproteinization
The soluble nucleic acids are re-extracted as described in Step 2 to remove any remaining proteins. This step may be repeated several times to assure complete removal of the proteins. Complete removal is assumed when very little protein is seen at the interface of the solvent layers.
6. Nucleic acid precipitation
Step 3 is repeated to purify further the nucleic acids.
7. Dissolution of nucleic acids
Step 4 is repeated to obtain a nucleic acid solution for further processing.
8. Ribonuclease treatment
The mixture of nucleic acids present in the solution is treated with ribonuclease for 30 minutes at 37.degree. C. to digest any RNA present in the sample. Following digestion, it is possible to remove proteins which were resistant to earlier extractions (steps 2 and 5).
9. Deproteinization
Step 2 is repeated to obtain DNA free of RNA, any proteins released by the ribonuclease treatment, and ribonuclease itself.
10. DNA precipitation
DNA is precipitated as described in step 3.
11. Dissolution of DNA
DNA is redissolved as described in step 4.
12. Isopropyl alcohol precipitation
To the DNA solution is added an acetate-EDTA buffer and the solution is mixed rapidly. While the solution is being mixed, 0.54 volume of isopropyl alcohol is added dropwise into the vortex. Then, according to the author, "DNA usually precipitates in a fibrous form after first going through a gel phase at about 0.5 vol(umes) isopropyl alcohol"--another difficult step.
13. Isopropyl alcohol precipitation
Steps 4 and 12 can be repeated "if the yield is good".
14. Final washing
The final precipitate is washed free of acetate and salt by gently stirring the adhered precipitate in aqueous ethanol containing progressively increasing (70-95%) portions of ethanol. The DNA is then available for dissolution in the buffer of choice for use in further analysis.
The process described by Marmur and followed to date by molecular biologists is complex and prone to loss of the desired DNA. According to Marmur, recovery of up to 50% of the DNA from the cells can be achieved by carefully following this process, not a very high yield. This process also generally requires 1 to 2 days, an undesirably lengthy processing period. Furthermore, according to Marmur, it would be very difficult to devise a technique for the efficient isolation of DNA from a wide variety of microorganisms. Although Marmur's method is effective against various organisms including almost all Gram negative organisms and many Gram positive organisms, one organism of great interest, Streptococcus pyogenes is not lysed by this method. This organism is the causative agent for the common illness, strep throat.
An alternative procedure for isolating DNA also disclosed by Marmur utilizes cesium chloride centrifugation. This process, while having many fewer steps, requires centrifugation for 3 days and, therefore, is also not a rapid method for isolating DNA. Additionally, cesium ions have a detrimental effect on the biological activity of the recovered DNA.
Carter et al. [Biotechniques, Volume 1(3), 142-147 (1983)] disclose an improved isopycnic centrifugation medium which uses cesium trifluoroacetate instead of cesium chloride. The cesium trifluoroacetate is used in a fashion similar to cesium chloride. The trifluoroacetate anion, however, imparts properties to cesium trifluoroacetate that result in higher quality nucleic acid preparations when compared to traditional cesium density gradient media. The use of cesium trifluoroacetate has extended the application of isopycnic centrifugation in nucleic acid separations and purifications but the procedure is still inherently time consuming and labor intensive.
Gross-Bellard et al. [European Journal of Biochemistry, Volume 36, 32-38 (1973)] disclose a similar method useful for isolating high molecular weight DNA from mammalian cells. In this method, Proteinase K and a detergent, sodium dodecyl sulfate (SDS), are used to lyse the cells. This method is also applicable to microorganisms susceptible to these lysis conditions. Deproteinization is accomplished using phenol saturated buffers rather than chloroform:isoamyl alcohol. The use of phenol, however, requires a 4-hour dialysis to remove it before proceeding further. Any RNA present is digested using ribonuclease and then the ribonuclease is digested using proteinase K and SDS. The DNA is then deproteinized twice more and dialyzed again. Finally, the DNA is precipitated with ethanol. This procedure offers little advantage over that of Marmur. There are fewer DNA precipitations, but the procedure introduces two long dialysis steps.
Chassy et al. [Applied and Environmental Microbiology, Volume 39(1), 153-158 (1980)] disclose a procedure for extending the usefulness of lysozyme in lysing microorganisms. This procedure depends upon growing the organism in a modified medium, particularly one containing L-threonine. This leads to organisms with weakened cell wall crosslinks which are susceptible to lysozyme treatment. This procedure is useful only when the organism to be lysed is known to grow in the modified medium and precludes any possibility of using DNA collected directly from a clinical specimen without culturing the organism. Therefore, this procedure is not of general utility.
Potter et al. [Cancer Letters, Volume 26, 335-341 (1985)] disclose a method for rapid extraction and purification of DNA from human leukocytes. This method includes detergent lysis of the cells, potassium acetate precipitation of cellular material, ribonuclease digestion, adsorption chromatography using DEAE-cellulose to purify the DNA, and ethanol precipitation of the DNA. This method is applicable only to those microorganisms susceptible to detergent lysis, generally the Gram negative organisms.
Potter et al.'s method is different from Marmur's in the use of potassium acetate to precipitate the cellular contents and the use of adsorption chromatography. These authors acknowledge that DNA can be lost in the potassium acetate precipitation step. Also, as with many precipitation methods, the sample must be centrifuged in order to assure complete recovery of the precipitate and centrifugation requires expensive equipment and valuable time. Avoidance of precipitation and centrifugation steps would be advantageous.
The use of adsorption chromatography to purify DNA can also have certain disadvantages since DNA with the highest molecular weight tends to bind most strongly to the support and, therefore, is not easily eluted from the column. This can result in selective loss of the highest molecular weight DNA.
De Klowet [Journal of Microbiological Methods, Volume 2, 189-196 (1984)] discloses a method for rapid isolation of high molecular weight RNA and DNA from yeast through the use of a single glucanase enzyme, lyticase, isolated from Oerskovia xanthineolytica, in the presence or absence of a detergent to lyse the cells. After lysing, the sample is deproteinized by extraction with an equal volume of a phenol:chloroform (4:1) solution. The mixture is centrifuged to separate the layers and the aqueous nucleic acid-containing phase is collected. This phase is made 0.3M in sodium acetate (pH 5.0) and the nucleic acids precipitated with two volumes of ethanol. Alternatively, high molecular weight RNA can be isolated by selective precipitation with lithium chloride. The high molecular weight DNA can be isolated from the supernatant of that precipitation or directly from the previously precipitated nucleic acids. In the former case, DNA isolation proceeds with ribonuclease treatment to destroy the RNA present. DNA is then deproteinized again by phenol:chloroform extraction and precipitated with ethanol. This overall procedure is very similar to that of Marmur in that it entails repeated deproteinization with organic solvents and repeated precipitation with ethanol, both of which are undesirable. These types of treatments are undesirable in that they require skilled technicians, extensive equipment and facilities and take substantial amounts of time.
Monsen et al. [FEMS Microbiology Letters, volume 16, 19-24 (1983)] disclose a general method for cell lysis and preparation of DNA from streptococci. This method uses the lytic enzyme mutanolysin (endo-N-acetylmuraminidase) isolated from Streptomyces globisporus 1829 to lyse the organism. As noted above, these organisms are resistant to lysozyme and detergent-induced lysis. Monsen et al. also showed that streptococci are resistant to a general protease, Proteinase K. High molecular weight DNA was isolated by Monsen et al. using cesium chloride centrifugation. As noted above, this is a very time consuming procedure not appropriate for routine preparation of clinical samples.
None of the methods discussed offers a completely general method for lysis of microorganisms of interest in clinical diagnostic applications and isolation of their DNA. In general, these methods utilize a single enzyme and/or a detergent to lyse a limited group of organisms.
Gillespie et al. (U.S. Pat. No. 4,483,920, issued Nov. 20, 1984) disclose the immobilization onto filters of messenger RNA in the presence of a high concentration (80%) of a chaotropic salt, sodium iodide. Here the chaotropic salt is used to denature proteins, to dissociate them from mRNA and to solubilize substantially all cellular components to allow them to pass through the hybridization filter.
Von Hippel et al., Science Volume 145, 577-580 (1964), studied the denaturation of proteins and nucleic acids with chaotropic salts and found that proteins are more susceptible to such denaturation than nucleic acids. One can conclude from such findings that it might be possible to select concentrations of chaotropic salts which will denature proteins, thus aiding their dissociation from nucleic acids, without denaturing double stranded high molecular weight DNA.
There remains a need for a rapid, efficient and simple process for isolating high molecular weight nucleic acids from a wide variety of sources.